7
Anal. Chem. 1095, 85, 7-13
Structure-Specific Collision=Induced Fragmentations of Ceramides Cationized with Alkali-Metal Ions Qinghong Ann and Jeanette Adams' Department of Chemistry, Emory University, Atlanta, Georgia 30322
Wllson-lnducod and metastable Ion fragmentatlons of gasphase [M Cat]+ complexes between the alkalhnetal Ions, LI+, Na+, K+, Rb+, and Cs+, and seven structurally dlverre ceramldes and two mhttures of ceramldes are reported. The cdlkkn-lnducod decomporltlon (CID) spectra of the [M U]+ Ions provldo Immodlate Information about the lengths of the sphlngoid base and fatty acyl chalns and locations of tubstltuentssuch as double bonds and hydroxylgroups on the fatty acyl chaln. Structures of lndlvldual components of complex mlxtures can be determlnd, and the detection llmk for structural detonnlnatlon of WpahnkoylsLaphlnganIneby C I D k 8 pmd. Rdatlveproductlon abundancw, which change wkh Increarlng rlze of a l k a l h t a l ion, cannot be explained by clarrlc klnetk arguments. They can be at least partlally explalnod, however, from docomposHlons of dMerent pow l a t h 8 of structurally dktlncl precursor ions.
+
+
INTRODUCTION Ceramides are a class of molecules that form the backbone of the biologically important sphingolipids. Ceramides (structure 1, R = H) comprise a long-chain sphingoid base, such as (11E)-sphingenine (R' = CH@H(CH2)12CH3) or OH R+. ' "
"
Y
R 0
structure I
sphinganine (R'= (CHz)&Ha), and a fatty acyl chain (R") substituted at the sphingoid amino group. Sphingolipids (R = sugar or other group in structure 1) are ubiquitous components of plasma membranes and are present at the outer leaflet of the membrane bilayer.' Sphingolipids can act as cell-surface interactants and transducers, cellular antigens, cell-surface markers, and as receptors for cellular recognition.2-4 Ceramides act both as precursors in the biosynthesis6 and aa intermediates in the degradations of sphingolipids. They also can be enzymatically hydrolyzed in vivo into their sphingoid base and fatty acid components.6 Because structures of ceramides can vary substantially along with their biological activity, their characterization has been of interest and a challenge in analytical chemistry. (1) Kanfer, J. N.; Hakomori, 5. SphingolipidBiochemistry,Handbook of Lipid Research; Plenum Press: New York, 1983; Vol. 3.
(2) Yogeeawaran, G.; Hakomori, S. Biochemistry 1976,14,2151-2156. (3) Ando, S.; Yamaknwa, T. J. Biochem. 1973, 73, 387-396. (4) Helting, T. B.; Zwisler, 0.;Wiegandt, H. J. Biol. Chem. 1977,252, 194-198. (5) Basu, 5.; Kaufman, B.; Roeeman, S. J.Biol. Chem. 1968,243,58025807. (6) Leibovitz, Z.; Gatt, 5. Biochim. Biophys. Acta 1968,152,136-143.
0003-2700/93/0365-0007$04.00/0
There have been several approaches for elucidating the structure of ceramides. One of the earliest methods employed chemical hydrolysis followed by derivatization and gas chromatography (GC) of the liberated sphingoid bases7 and fatty acids.8~9This approach for structure determination of the sphingoid bases was later improved by the addition of electron ionization (EI)GC-mass spectrometry (GC-MS).'O Samuelsson and Samuelsson,"J2 however, were the fmt to determine ceramides as intact molecules by derivatization to trimethylsilyl ethers followed by GC-MS. They and others used GC-MS of the trimethylsilyl ethereto identify ceramides that contained a-hydroxyfatty acyl groups1*'6 and ones that contained phytosphingosine (4-~-hydroxysphinganine).~61~~ Similar approaches involved chemical ionization of acetylated ceramidesl6 or E1 of permethylated species.19~~ Fast atom bombardment (FAB) MS of both [M + HI and [M - HI-ions has provided a new avenue for determiningthe molecular weights of ceramides released from sphingolipids.21-24 Unfortunately, interfering matrix ions and poor sensitivity associated with detection of fragment ions precludes unequivocal structural characterization by FAB alone. High-energy (kiloelectronvolt) tandem mass spectrometry (MS-MS) can alleviate some of these problems, but the exact location of substituents such as double bonds in the N-acyl chain cannot be determined.*% Costello and co-workers, however, have demonstrated that either permethylation or hydroboration and oxidation of intact ceramides coupled with MS-MS of the [M + HI+ ions could be used to determine their structures.27~28 +
(7) Karlsson, K.; Holm, G. A. L. Acta Chem. Scand. 1966,19,24232425. (8) Sweeley, C. C. J. Lipid Res. 1963,4, 402-406. (9) Morrison, W. R. Biochim. Biophys. Acta 1969,176,537-646. (10) Polito, A. J.;Akita, T.; Sweeley, C. C. Biochemistry 1968,7,260% 2614. (11) Samuelsson, B.; Samuelsson, K. Biochim. Biophys. Acta 1968, 164,421-423. (12) Samuelseon, B.; Samuelsson, K. J. Lipid Res. 1969, 10, 41-48. (13) Samuelsson, K.; Samuelsson, B. Chem. Phys. Lipids 1970,5,4479 and references therein. (14) HammarstrBm, S.; Samuelsson, B.; Samuelsson, K. J. Lipid Res. 1970,11,150-157. (15) HammarstrBm, S. Eur. J. Biochem. 1970,15,581-591. (16) Hayashi, A,; Matauura, F. Chem. Phys. Lipids 1973, 10, 51-65. (17) HammamtrBm, 5. J. Lipid Res. 1970,11, 175-182. (18) Markey, S. P.; Wenger, D. A. Chem. Phys. Lipids 1974,12,182200. (19) Egge, H. Chem. Phys. Lipids 1978,21, 34!t360. (20) Hansson, G.; Li, Y. T.; Karlsson, H. Biochemistry 1989,28,&3726678. (21) Hemling, M. E.; Robert,K.; Yu, R.; Sedgwick, D.; Rinehart, K. L. Biochemistry 1984,23,5706-5713. (22) PBhleson, P.; Nilsson, B.Anal. Biochem. 1988,168,115-120. (23) Suzuki, M.; Seine, M.; Yamakawa, T.; Suzuki, A. J. Biochem. 1989, 105, 829-833. (24) Suzuki, M.;Yamakawa, T.; Suzuki, A. J. Biochem. 1990,108,9298. (25) Singh, B. N.; Costello, C. E.; Levery, S. B.;Walenga, R. W.; Beach, D. H.; Mueller, J. F.; Holz, G. G. Mol. Biochem. Parasitol. 1987,26,99112. (26) Domon, B.; Costello, C. E. Biochemistry 1988,27,1534-1543. (27) Domon, B.; Vath, J. E.; Costello,C. E. Anal. Biochem. 1990,184, 151-164. (28) Costello,C. E.; Vath, J. E. InMethods in Enzymology;McCloekey, J. A., Ed.; Academic Press: San Diego, CA, 1990; Vol. 193 pp 738-768. Q 1993 American Chemical Sockty
8
ANALYTICAL CHEMISTRY, VOL. 65, NO. 1, JANUARY 1, 1993
As an alternative to chemical derivatization, we have been exploring the use of metal ions as adducts to direct mass spectral fragmentation pathways to improve structural determination. Earliest work involved collision-induced decomposition (CID) of FAB-desorbed [M+ Cat]+ or [M+ 2Cat - H]+ ions, in which Cat = alkali-metal ion, to determine structures of fatty alcohols and acids.2*32 This approach has since been applied to structure determination of prostaglandins,33fatty acid esters,34 and peptides.3"37 Others have likewise used this approach to elucidate structures of sugars and oligosaccharides3~2and other compounds.43~" Preliminary data were recently presented for structure determination of ceramides and neutral glycosphingolipids by CID of [M+ Lil+ i0ns.~6 Here we report in more detail the use of this technique for determining structures of intact ceramides.
vvv\
O
K
(M+H)+
t . ,. ,.t 388
. 158
10E
358
EXPERIMENTAL SECTION Reagents. Seven ceramides and two mixtures of ceramides (Sigma, St. Louis, MO) were used in this investigation. The ceramides are N-lignoceroyl-DL-sphinganine(N-tetracosanoylDL-Sphinganine),N-palmiotyl-DL-sphinganine(N-hexadecanoylDL-sphinganine),N-stearoyl-DL-sphinganine(N-octadecanoylDL-sphinganine),N-palmitoyl-(4E)-sphingenine(N-hexadecanoyl(4E)-sphingenine), N-oleoyl-(4E)-sphingenine(N-cis-9-octadecenoyl-(4E)-sphingenine),N-nervonoyl-(4E)-sphingenine (Ncis-15-tetracosenoyl-(4E)-sphingenine),and N-stearoyL(4E)sphingenine (N-octadecanoyl-(4E)-sphingenine).One of the mixtures, ceramide type IV, which contains a-hydroxy-Nlignoceroyl-(4E)-sphingenine as a dominant component,@was used to study ceramides that contain an a-hydroxy fatty acyl chain. Another mixture, ceramide type 111,was used for mixture analysis. Procedure. The sampleswere dissolved in CHCldMeOH (2:l v/v), and 1 pL of this solution was added to the FAB matrix on the stainless-steel FAB probe tip. The [M+ HI+ and [M HI- ions were desorbed from a matrix of 3-nitrobenzyl alcohol (3-NBA). The [M + Cat]+ complexes of most ceramides were prepared by using 3-NBA saturated with different alkali-metal iodides. Complexes of the a-hydroxy fatty acyl ceramides were instead prepared by using 3-NBA saturated with alkali-metal hydroxides. Deuterium-labeled a-hydroxy-N-lignoceroyl-(4E)sphingenine-d4was desorbed as [M + Li]+ ions by dissolving the ceramide in CDCldMeOD (2:1, v/v) and then adding - 1 pL of this solution to deuterium-exchanged 3-NBA, saturated with LiOD and D20.The deuterium-exchanged 3-NBAwas prepared by dissolving it in D20 and then evaporating the DzO under vacuum four times. Mass spectrometric experiments were conducted by using a VG 7 0 4forward-geometry(EB,where E is electrostatic analyzer, or ESA, and B is magneticanalyzer) mass spectrometer equipped
-
(29) Adams, J.; Gross, M. L. J. Am. Chem. SOC.1986,108,6915-6921. (30) Adams, J.; Gross, M. L. Anal. Chem. 1987,59, 15761582. (31) Adams, J.; Gross, M. L. Org. Mms Spectrom. 1988,23,307-316. (32) Adams, J.; Deterding, L. J.; Gross, M. L. Spectroscopy (Ottawa) 1987,5, 199-228. (33) Contado, M. J.; Adams, J.; Gross, M. L. Adu. Mass Spectrom. 1989,11B, 1034-1035. (34) Contado, M. J.; Adams, J. Anal. Chim. Acta 1991,246,187-197. (35) Teesch,L.M.;Adams,J. J.Am. Chem.Soc. 1990,112,4110-4120. (36) Teesch, L. M.; Adams, J. J. Am. Chem. SOC.1991,113,812-820. (37) Tsesch, L. M.; Orlando, R. C.;Adams, J. J. Am. Chem. SOC.1991, 113,3668-3675. (38) Wright,L. G.; Cooks,R. G.;Wood, K. V. Biomed. Mass Spectrom. 1985,12, 159-162. (39) Orlando, R.; Bush, C. A.; Fenselau, C. Biomed. Enuiron. Mass Spectrom. 1990,19,747-754. (40) Zhou, Z.; Ogden, S.; Leary, J. A. J . Org. Chem. 1990, 55, 54445446. (41) Hofmeister, G. E.; Zhao, Z.; Leary, J. A. J.Am. Chem. SOC.1991, 113,5964-5970. (42) Puzo, G.; Prome, J. C. Adu. Mass Spectrom. 1979,8,1003-1011. (43) Teesch, L. M.; Adams, J. Org. Mass Spectrom., in press. (44) Teesch, L. M.; Adams, J. In Experimental Mass Spectrometry; Russell, D. H., Ed.; Plenum Press (in press). (45) Ann, Q.; Adams, J. J. Am. SOC.Mass Spectrom. 1992,3,260-263. (46) Carter, H. E.; Rothfus, J. A.; Gigg, R. J. Lipid Res. 1961,2,228234.
(M+Li)+
402
258
388
He
(80
mlz
+
430
458
SEE
+
-
Flgurr 1. CID spectra of (a) [M HI+, (b) [M H]-, and (c) [M U]' ions of Mpaimitoyy4&sphingenhe. See Flgve2 for nomendatwe for the cleavages. In all spectra, the masses of the precursor and product Ions are shown without thelr posithre mass defects (e.g., mlr 538 Is actually mlz 538.5).
with an Ion Tech saddle-field FAB gun and a commercial FAB ion source. Precursor ions were produced by bombarding the sample with 7-keV Ar or Xe atoms at a gun current of 2 mA. Sample ions desorbed from the FAB matrix were accelerated to 8-keV translational energy, and product ions formed either metastably or by CID in the first field-free region between the ion source and ESA were observed by using scans at a constant ratio of B/E. Helium was used as collision gas, and the beam transmission was reduced to -50%. An aqueous solution of LiI (1.5F),NaI(4.5F),KI(0.01F),Rb1(0.01F),andCsI(1.0F) was prepared and mixed 5:l (v/v) with acetonitrile for calibration." Experimentswere performedat a product ion resolutionof l o o 0 ( 1 0 % valley), and 10-15 scans were signal-averaged,smoothed, centroided, and mass-measured by using commercial software. Backgroundsubtraction of matrix and other spectra was acquired for all experiments, and this was especially important in determining the detection limit and performing the mixture analysis."
-
RESULTS AND DISCUSSION Structural Determination. In an earlier communication,45 we reported that CID of [M + Lil+ ions of ceramides provides more structural information than CID of either [M + HI+ or [M - HI-ions. Examples of the type of structural information that can be obtained from CID of the three different precursor ions are shown in Figure 1for N-palmitoyl(a)-sphingenine. The nomenclature used to label the peaks in the spectra is defiied in Figure 2. Loss of water is one major fragmentation route for CM + HI+ions of ceramides that contain both (rlE)-sphingenine (Figure la) and sphinganine. There are also 0' and 0"ions that presumably arise (47) Sato,K.;Asada,T.;Ishihara,M.;Kunihiuo,F.;Kammei,Y.; Kubota, E.; Costello, C. E.; Martin, S. A.; Scoble, H. A.; Biemann, K. Anal. Chem. 1987,59, 1652-1659.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 1, JANUARY 1, 1893
0
cleavages of ceramides coniain sphingoid chain J, K,'O, P
contain N-acyl chain H, S,T, U, V
P
OH
r-. ,
H
Flgurr 2. Nomenclaturefor cleavages of precursor Ions of ceramkies.
Scheme I 0
O
